Compositions to promote the healing of skin ulcers and wounds

ABSTRACT

The present invention provides compositions comprising as an essential feature granulocyte-macrophage colony-stimulating factor (GM-CSF) together with fosfomycin for the treatment of wounds, ulcers, sores, burns and other injuries to the skin or mucous membranes of the body.

INCORPORATION BY REFERENCE TO ANY PRIORITY APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/596,233 currently pending, filed on Oct. 8, 2019, which is acontinuation of U.S. patent application Ser. No. 15/116,767, filed onAug. 4, 2016, which is a U.S. National Phase Application of PCTInternational Application Number PCT/EP2015/052411, filed on Feb. 5,2015, designating the United States of America and published in theEnglish language, which is an International Application of and claimsthe benefit of priority to Danish Patent Application No. PA 2014 70059,filed on Feb. 5, 2014. The disclosures of the above-referencedapplications are hereby expressly incorporated by reference in theirentireties.

REFERENCE TO SEQUENCE LISTING

A Sequence Listing submitted as an ASCII text file via EFS-Web is herebyincorporated by reference in accordance with 35 U.S.C. § 1.52 (e). Thename of the ASCII text file for the Sequence Listing isSeqList-ZACCO108-003C2.txt, the date of creation of the ASCII text fileis Aug. 8, 2021, and the size of the ASCII text file is 3 KB.

BACKGROUND OF THE INVENTION Field of the Invention

The present invention provides compositions comprising as theiressential ingredients granulocyte-macrophage colony-stimulating factor(GM-CSF) and the antibiotic fosfomycin, for the treatment of wounds,ulcers, sores, burns and other injuries of the skin or membranes of thebody. As such, it is relevant to the fields of dermatology and nursingcare in the areas of medicine and surgery, traumatology and burns.

Background of the Invention Wound Healing

Wound healing is a normal feature of living animals and is a dynamic,interactive process which involves various types of cell and theextracellular matrix, depending for its speed and efficiency on variousinternal and external factors. The normal healing process can bedescribed in terms of four programmed phases comprising 1) hemostasis,2) inflammation, 3) proliferation and 4) remodeling.

-   -   1) Hemostasis is the vascular response stage that occurs        immediately after the insult and normally lasts for up to a few        hours. The wound may bleed initially, but blood clotting and        vasoconstriction re-establish hemostasis while coagulated blood        provides a provisional extracellular matrix for cell migration.    -   2) Inflammation normally starts at the time of injury and        finishes within 3-4 days in the course of normal healing. The        inflammation phase is clinically recognized by the classical        signs and symptoms of inflammation, such as heat, redness,        swelling and pain. The wound starts to exude fluid, which serves        to remove debris, and proteases are released into the wound        area. White blood cells and macrophages begin to congregate in        the lesion to clear debris: Growth factors released by the        macrophages stimulate fibroblasts. During this phase the        extracellular matrix is constructed. Inflammation is always        present, while the entry of bacteria may pass from colonization        to clinical infection to exacerbate any delay in the resolution        of the inflammatory phase.    -   3) Proliferation normally continues after the inflammatory phase        and is characterized by the proliferation and migration of        fibroblasts and the production of connective tissue. It starts        about 4 days after the occurrence of the trauma and continues        for 2-3 weeks in a normally healing closed wound. This phase can        be extended significantly in an open wound with severe tissue        damage, where complete closure will require the production of a        large amount of connective tissue. During this phase,        neovascularization, epithelialization, wound contraction and        collagen production are taking place.    -   4) Remodeling (also called maturation, moderation or scar phase)        starts 2-3 weeks after wound closure, while it does not start in        open wounds before the wound has healed. It typically continues        for several weeks, months or even years thereafter. Maturation        involves contraction of the wound, growth of new epithelial        tissue covering the granulation tissue, and possibly scar        formation. During this phase myofibroblasts develop from the        fibroblasts and the collagen fibers gradually mature and become        relatively more organized.

For a wound to heal optimally, all four phases must occur in the propersequence and timeframe. Different parts of a wound may heal at differentrates, so that some parts of a normal wound may be at a more advancedstage of healing than others.

Delayed Wound Healing and Chronic or Non-Healing Wounds and Ulcers

Many factors may cause abnormal or impaired wound healing includingprolonged failure to heal (non-healing wounds). Such wounds havegenerally failed to progress through the normal stages of healing andoften enter a state of pathologic inflammation due to the delayed,incomplete, or uncoordinated healing process. These wounds are typicallycolonized or infected by one or more species of bacteria whichexacerbate and prolong the inflammation and contribute to the delay inhealing.

Most chronic wounds are ulcers that are associated with ischemia,diabetes mellitus, venous stasis or pressure. Non-healing wounds affectmore than 1% of the industrialized world's population. In the UnitedStates they may affect up to 6 million people, about 85% of whom will beaged with persons 65 years or and older. Non-healing wounds result inenormous health care expenditures, with a total annual cost estimated atmore than $3 billion in the United States.

The prevalence is expected to increase as the population ages and thenumber of individuals with diabetes mellitus increases. Chronic ulcersreduce the quality of life and working capacity of the patient andrepresent a substantial financial burden to the health care system.

Ulcers of the lower extremities, especially those attributed to diabetesmellitus, or venous or arterial insufficiency, comprise a substantialproportion of chronic ulcers. Approximately 15% to 25% of individualswith diabetes mellitus develop a foot ulcer at some point in theirlifetime and an estimated 12% of those patients require lower extremityamputation. Healing is complicated by diabetic neuropathy andsusceptibility to infection is greatly increased. Venous diseaseaccounts for the majority of chronic lower extremity ulcers. Venoushypertension secondary to various causes can damage the blood vesselwalls and ultimately leads to skin breakdown. Arterial ulcers are lesscommon and are a result of impaired circulation which can adverselyaffect healing and lead to ulceration.

Role of Bacterial Colonization and Infection in Chronic Ulcers

In the inflammation phase of impaired wound healing, bacteria willalways contaminate the ulcer in a process passing through colonizationand critical colonization to infection. Critical colonization is notalways associated with overt signs of infection but can result infailure to heal, poor-quality granulation tissue, increased woundfriability, and increased exudation (Frank C et al., 2005). Themicroflora of chronic wounds such as ulcers most commonly exist in thebiofilm phenotype and have been known to significantly impair normalhealing trajectories (Smith D M et al., 2010). Standard treatmentinclude the use of topical antimicrobials (iodine and silverpreparations) or topical antibiotics (Mupirocin, fusidic acid,aminoglycosides, bacitracin, polymyxin B, gramicidins and metronidazole,alone or in various combinations) for two weeks or so.

This May be Followed by Giving Relevant Oral Antibiotics.

Types of bacteria involved in chronic skin ulcers The types of bacteriainvolved in colonization and infection of chronic skin ulcers typicallyinclude those that are present in the contralateral or surroundingintact skin, but will show an greatly enhanced presence of certainbacterial genera, including, for example, Corynebacterium, Pseudomonas,Streptococcus, Escherichia, Shigella and Serratia in a study of diabeticulcers (Gontcharova V et al., 2010). Anaerobic genera such as Finegoldiaand Peptoniphilus are also common. While Staphylococcus was notoverrepresented as a genus, infection of chronic wounds with S. aureusmust be taken seriously. In an analysis of decubitus ulcers, the mostcommon bacterial genera identified were Streptococcus, Corynebacterium,Staphylococcus, Finegoldia, Anaerococcus, Pseudomonas and Peptoniphilus.Many functionally equivalent pathogroups (symbiotic colonies ofotherwise nonpathogenic species that act synergistically to promotetheir own survival at the expense of the host), were found to colonizeand exist as a chronic wound pathogenic biofilm, which is accepted to bethe primary infectious agent in chronic wounds (Smith D M et al., 2010).

Standard Treatment of Non-Healing Wounds and Ulcers

Standard treatment for all non-healing wounds and ulcers includesdebridement of necrotic tissue, infection control and local wound care.For each category of non-healing wound, specialized treatment modalitieshave to be implemented. For diabetic foot ulcers, for example, attentionhas to be paid to mechanical off-loading, tight blood glucose controland patient education on foot care. For venous ulcers, special measurestypically include mechanical compression and limb elevation to reversetissue edema and improve venous blood flow. Care for ulcers caused byarterial insufficiency is centered on reestablishing blood flow andminimizing further loss of tissue perfusion. For pressure ulcers,off-loading methods or devices are the gold standard of treatment.

If ulcers do not adequately heal with standard treatment, additionaltreatment modalities may be required, which are often termed “advancedwound care therapies.” A large and growing array of advanced wound caretherapies of different composition and indications have been developed.For a considerable number of these therapies their efficacy, comparativeeffectiveness and adverse effects are not well established. Thus,although there has been extensive research in the field of wound healingtreatment, the healing of wounds and ulcers is still a complex task,particularly in the elderly or diseased population.

Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF)

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a member ofthe family of colony-stimulating factors (CSFs), which are glycoproteinsthat stimulate the proliferation and maturation of hematopoieticprogenitors and enhance the functional activity of mature effectorcells. In brief, at the level of the immature cells, CSF's ensure theself-renewal of the staminal pool and activate the first stage ofhematopoietic differentiation. In the subsequent stage, when cellproliferation is associated with a progressive acquisition of thecharacteristics of the mature cells, they enormously enhance the numberof differentiating cells. In the terminal stage, they stimulate thecirculation and the activation of mature cells.

GM-CSF has been shown to have a positive effect on wound healing byfacilitating wound contraction, causing local recruitment ofinflammatory cells, and inducing keratinocyte proliferation. GM-CSF alsoactivates mononuclear phagocytes, promotes migration of epithelialcells, and further regulates cytokine production in the healing process.

Mature GM-CSF is a monomeric protein of 127 amino-acid residues withseveral potential glycosylation sites. The variable degree ofglycosylation results in a molecular weight range between 14 kDa and 35kDa. Non-glycosylated and glycosylated GM-CSF show similar activity invitro (Cebon J et al., 1990). The crystallographic analysis of GM-CSFrevealed a barrel-shaped structure composed of four short alpha helices(Diederichs K et al., 1991). There are two known sequence variants ofGM-CSF. The active form of the GM-CSF protein is found extracellularlyas a homodimer in vivo.

GM-CSF exerts its biological activity by binding to its receptor. Themost important sites of GM-CSF receptor (GM-CSF-R) expression are on thecell surface of myeloid cells, such as macrophages types I and II,epithelial cells and endothelial cells, whereas lymphocytes are GM-CSF-Rnegative. The native receptor is composed of alpha and beta subunits.The alpha subunit imparts ligand specificity and binds GM-CSF withnanomolar affinity. The beta subunit is also part of the interleukin-3and interleukin-5 receptor complexes and, in association with theGM-CSF-R alpha subunit and GM-CSF, leads to the formation of a complexwith picomolar binding affinity (Hayashida K et al., 1990). The bindingdomains on GM-CSF for the receptor have been mapped: GM-CSF interactswith the beta subunit of its receptor via a very restricted region inthe first alpha helix of GM-CSF (Shanafelt A B et al., 1991 a; b; LopezA F et al., 1991). Binding to the alpha subunit could be mapped to thethird alpha helix, helix C, the initial residues of the loop joininghelices C and D, and to the carboxyterminal tail of GM-CSF (Brown C B etal., 1994).

Formation of the GM-CSF trimeric receptor complex leads to theactivation of complex signaling cascades involving molecules of theJAK/STAT families, She, Ras, Raf, the MAP kinases,phosphatidylinositol-3-kinase and NFkB, finally leading to thetranscription of c-myc, c-fos and c-jun. Activation is mainly induced bythe beta subunit of the receptor (Hayashida K et al., 1990; Kitamura Tet al., 1991; Sato N et al., 1993). The shared beta subunit is alsoresponsible for the overlapping functions exerted by IL-3, IL-5 andGM-CSF (reviewed by de Groot R P et al., 1998).

In addition to its stimulating activity on hemopoietic growth anddifferentiation, GM-CSF acts as a proinflammatory cytokine. Macrophages,e.g. macrophages type I & II and monocytes, as well as neutrophils andeosinophils, are activated by GM-CSF, resulting in the release of othercytokines and chemokines and matrix-degrading proteases, as well asincreased expression of HLA and cell adhesion molecules or receptors forCC-chemokines. This in turn leads to increased chemotaxis ofinflammatory cells into inflamed tissue.

Macrophages in wounds are known to release a variety of biologicallyactive substances that serve as chemo-attractants for both monocytes andfibroblasts, such as transforming growth factor-β (TGF-β) andplatelet-derived growth factor (PDGF). Activated macrophages digestdevitalized collagen and the fibrin clot. Dissolution of the clot allowsthe formation of granulation tissue in the wound site during the secondphase of wound healing.

Use of GM-CSF to Stimulate Wound Healing

The activities of GM-CSF outlined above not only stimulate the cellularproliferation, maturation and activities that are involved in theinflammation and proliferation stages of wound healing, but also, by thesame processes, enhance the innate defense mechanisms against bacterialinfection. The use of topically applied recombinant human GM-CSF toaccelerate the healing of various types of chronic wounds and ulcers hasbeen systematically reviewed and concluded to be beneficial for deeppartial-thickness burns, chronic leg ulcers and leprosy ulcers, withsome evidence for a positive effect on pressure ulcers andcancer-related ulcers (Hu X et al., 2011). Topically applied GM-CSF toexperimental wounds infected with Escherichia coli in rats was found tostimulate the innate defense against infection as seen by reducedbacterial counts in association with accelerated wound closure (Robson Met al., 1994).

The Use of Topical Antibiotics to Control Bacterial Infection in ChronicWounds and Ulcers

The current use of topical microbial agents and antibiotics in thestandard care of chronic wounds and ulcers has been outlined above.Topical antimicrobials such as preparations based on iodine or silvermay be likened to antiseptics in general: while they may have powerfulbactericidal action, they also exert significant toxic effects on theliving cells that are essential for the process of wound healing. Thebalance between the local bactericidal effect and the toxicity to thehost tissue is an important consideration. This balance may be morefavorable for the promotion of healing in the case of the antibioticsmentioned, but these antibiotics may also have significant cytotoxicactions at the local concentrations reached. It is evidentlyadvantageous to choose an antibiotic or combination of antibiotics thatis strongly bactericidal while having little or no toxicity towards thecells involved in wound healing. A further consideration is that theantibiotic should be active against the principal bacterial species thatare known to occur in infected wounds and ulcers. In addition, it is agreat advantage if the antibiotic is currently active against manymulti-drug resistant bacterial strains that have become more prevalentin recent years. Finally, it is an advantage if there has been noappreciable increase in bacterial resistance to the drug over anextended period of time, so that there is a reasonable expectation thatbacterial resistance to it will be

Fosfomycin

Fosfomycin is the international non-proprietary name of a broad-spectrumantibiotic isolated and characterized in 1969 from Streptomycesfradiaestrains under the name phosphomycin or phosphonomycin (Hendlin D et al.,1969). Its structure was determined to be (−)(IR,2S)-1,2-epoxypropylphosphonic acid (Christensen B G et al., 1969), withthe systematic (IUPAC) name [(2R,3S)-3-methyloxiran-2-yl]phosphonic acidand a formula weight of 138.1 Da. Fosfomycin is bactericidal andinhibits bacterial cell wall biosynthesis by inactivating the enzymeUDP-N-acetylglucosamine-3-enolpyruvyltransferase, also known as MurA(Brown E D et al., 1995). This enzyme catalyzes the committed step inpeptidoglycan biosynthesis, the ligation of phosphoenolpyruvate to the3′-hydroxyl group of UDP-N-acetylglucosamine to form N-acetylmuramicacid. Fosfomycin is a phosphoenolpyruvate analogue that inhibits MurA byalkylating an active site cysteine residue. The antibiotic enters thebacterial cell via the glycerophosphate transporter.

Given this mechanism of action, fosfomycin has a broad bactericidalspectrum, being active against aerobic genera such as Staphylococcus,Streptococcus, Neisseria, Escherichia, Proteus (in dole-negative),Serratia, Salmonella, Shigella, Pseudomonas, Haemophilus, and Vibrio,less active against indole-positive Proteus spp., Klebsiella andEnterobacter spp. It is known to be active against the anaerobic generaPeptostreptococcus (including Peptoniphilus, Finegoldia andAnaerococcus) and Fusobacterium. It will be seen that this spectrum ofactivity comprises a large number of the genera prominent in criticalcolonization and infections of chronic skin ulcers.

There is a low prevalence of bacterial resistance to fosfomycin in thecommunity, and studies of the prevalence of resistant bacteria after theintroduction of fosfomycin have shown either no increase or only amodest increase in the prevalence of resistant organisms. However,prolonged exposure to the antibiotic may enable bacteria to evolveresistance by selection of mutants that lack the glycerophosphatetransporter pathway. Alternative mechanisms of resistance involve theloss of the inducible hexose phosphate transporter, a Cys-Asp mutationin MurAS, or acquisition of plasmids coding for the fosfomycininactivating enzymes fosA and fosB (in addition to the chromosomal fosXin Listeria monocytogenes). The mutant strains may, however, also showreduced pathogenicity (Karageorgopoulos D E et al., 2012). This mayexplain why the emergence of bacterial resistance is seen on prolongedexposure in vitro, but much less frequently in vivo. The appearance ofresistant bacterial strains in controlled clinical trials of orally orintravenously administered fosfomycin has been 3.0% overall, with amaximum of 15% for Pseudomonas aeruginosa. In general, fosfomycin isseen to be a valuable addition to the therapeutic armament againstmultidrug-resistant organisms. There is no experience with thedevelopment of resistance to fosfomycin during treatment of bacteriallyinfected chronic ulcers.

Fosfomycin has proved to be remarkably non-toxic to mammalian cells andorgans, despite fosfomycin disodium being used at intravenous doses ofup to 0.5 g/kg/day in human patients. Here the limiting factor isoverload with the counter-ion rather than any toxic effect of theantibiotic. Indeed, fosfomycin has been found to exert a protectiveeffect against the toxic action of other antibiotics, immunosuppressiveor chemotherapeutic agents such as aminoglycosides, vancomycin,amphotericin B, polymyxin, ciclosporin and cisplatin (Gobernado M,2003). As additional effects it has the capacity to favor phagocytosisand act as an immunomodulator. It is accumulated by polymorphonuclearleukocytes to reach concentrations that are twice those of theextracellular fluid, but does not affect their cellular functions, whileexerting a bactericidal effect on Staphylococcus aureus. The chiefadverse effects are gastric irritation from orally administeredfosfomycin disodium, evidence of allergy in the form of transient rashes(0.3% of cases) and eosinophilia (0.2%), and transiently raised liverenzymes (0.3% of cases) (Gobernado M, 2003).

The Use of Fosfomycin in Combination with Other Antibiotics

Fosfomycin shows a considerable synergism in bactericidal effect on alarge number of strains of organisms from the susceptible generamentioned, when used in combination with a large number of antibioticsof the penicillin, cephalosporin, aminoglycoside, macrolide andlincosamide types. While early studies showed a synergistic effect onabout 70-100% of tested strains for various antibiotic combinations,subsequent more extensive studies showed synergy rates of 36-74%. Theremaining strains showed merely additive effects and an inhibitoryeffect was only seen in one or two individual antibiotic combinations onan individual bacterial strain (Gobernado M, 2003). The fact thatfosfomycin shows synergy with many individual antibiotics and indeedabrogates the toxicity of many other antibiotics, including thenephrotoxicity and ototoxicity of the aminoglycosides, favors the use offosfomycin in combination with other antibiotics to produce a potentbactericidal action and compensate for any development of fosfomycinresistance during more prolonged treatment.

SUMMARY OF THE INVENTION

Accordingly, the present invention provides pharmaceutical compositionsfor the topical treatment of wounds, chronic ulcers and sores or theskin and mucous membranes that show delayed healing or non-healing andare critically colonized or infected with bacteria, said compositionscomprising essentially

-   -   1. A pharmaceutical composition comprising        -   a) granulocyte-macrophage colony-stimulating factor (GM-CSF)            or a fragment or variant thereof, and        -   b) fosfomycin in the form of an inorganic or organic salt            thereof.    -   2. A pharmaceutical composition according to 1. above,        comprising one or more additional antibiotic or antimicrobial        agents.

The advantage of combining GM-CSF with fosfomycin or fosfomycin incombination with other antibiotics is to exploit the known effect ofGM-CSF to promote wound healing and the innate defense mechanismsagainst bacterial infection while further accelerating wound healing bymore rapidly eliminating the infecting bacteria through the bactericidalaction of fosfomycin, which has no toxic action on the granulocytes,macrophages and other cells through which the healing effect of GM-CSFis exerted. The effect is to achieve a faster rate of healing of woundsand ulcers showing delayed healing or non-healing than that achieved byexisting treatments or by the use of either topical GM-CSF or topicalfosfomycin, with or without an additional antibiotic, alone.

In the following detailed description of the invention, details of thescope of the invention and the meaning of the terms used will be given,together with details of the practical performance of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

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DETAILED DESCRIPTION OF THE INVENTION

The present invention provides compositions comprisinggranulocyte-macrophage-colony stimulating factor (GM-CSF) or a fragmentor variant thereof, fosfomycin in the form of an inorganic or organicsalt thereof, and in a further embodiment, an additional antibiotic orantimicrobial agent, and in a still further embodiment, an additionalagent which has been found to promote wound healing. Such compositionsare useful for the treatment of wounds, ulcers, sores and other types ofinjury to the skin and mucous membranes and are intended to be topicallyapplied in various ways which be further described. The compositions andmethods of the present invention are also in some embodiments useful inmethods of treatment.

GM-CSF Preparations

For practical purposes, the GM-CSF preparations to be used in thepresent invention will not be purified native human GM-CSF, which couldof course be used if it were available in sufficient quantity andproblems of possible viral contamination were overcome, but human GM-CSFprepared in vitro by recombinant DNA technology. The preparation ofhuman recombinant GM-CSF (hrGM-CSF) in mammalian cells has beendescribed (Wong G G et al., 1985; Kaushansky K et al., 1986). Similarwork has led to the production of hrGM-CSF with the non-proprietary nameregramostim in Chinese hamster ovarian (CHO) cells (first reported byMoonen P et al., 1987). The expression of hrGM-CSF in Saccharomycescerevisiae was reported by Cantrell M A et al. (1985), leading to thepreparation known by the non-proprietary name sargramostim. Sargramostimdiffers from endogenous human GM-CSF in having a leucine residue insteadof a praline residue at position 23 of the pro-peptide and is lessglycosylated than either endogenous human GM-CSF or regramostim(Armitage J O, 1998). The expression of hrGM-CSF in Escherichia coli wasreported by Burgess A W et al. (1987), leading to the preparation knownby the non-proprietary name molgramostim, which is not glycosylated. Allthree hrGM-CSF preparations, regramostim, sargramostim and molgramostimcan be used in the present invention, but only the last two arecurrently available.

A “functional homologue” of human GM-CSF is herein defined as apolypeptide having at least 50% sequence identity with the known andnaturally occurring sequence and sequence variants of human GM-CSF andhas one or more functions of the naturally occurring protein. Thesefunctions include the following: stimulating the growth anddifferentiation of hematopoietic precursor cells from various lineages,including granulocytes, macrophages and monocytes, enhancing functionalactivities of mature effector cells involved in antigen presentation andcell-mediated immunity, including neutrophils, monocytes, macrophages,and dendritic cells. The functions also include those of particularrelevance to wound healing, such as facilitating wound contraction,causing local recruitment of inflammatory cells, improving therecruitment of neutrophils, inducing keratinocyte proliferation,activating mononuclear phagocytes, promoting the migration of epithelialcells, and further regulating cytokine production in the healingprocess. Regramostim, sargramostim and molgramostim may all be said tobe functional homologues of naturally occurring human GM-CSF.

Evolutionary conservation between GM-CSF homologues of different closelyrelated species, as assessed by amino-acid sequence alignment, can beused to pinpoint the degree of evolutionary pressure on individualamino-acid residues. Preferably, GM-CSF sequences are compared betweenspecies where GM-CSF function is conserved, for example, but not limitedto mammals, including rodents, monkeys and apes. Residues under highselective pressure are more likely to represent essential amino acidresidues that cannot easily be substituted than residues that changebetween species. It is evident from the above that a reasonable numberof modifications or alterations of the human GM-CSF sequence can be madewithout interfering with the activity of the GM-CSF molecule accordingto the invention. Such GM-CSF molecules are herein referred to asfunctional homologues of human GM-CSF, and may be such variants andfragments of native human GM-CSF as described below.

As used herein, the expression “variant” refers to a polypeptide orprotein which is homologous to the index protein, which is naturallyoccurring human GM-CSF in the present instance, but which differs fromthe index protein in that one or more amino-acid residues within thesequence of the index protein are substituted by other amino-acidresidues. These substitutions may be regarded as “conservative” when anamino-acid residue is replaced by a different amino-acid residue withbroadly similar properties, and “non-conservative” when an amino-acidresidue is replaced by one of a different type. Broadly speaking, fewernon-conservative substitutions will be possible without altering thebiological activity of the polypeptide.

A person skilled in the art will know how to make and assess“conservative” amino-acid substitutions, by which one amino-acid residueis substituted by another having one or more shared chemical and/orphysical characteristics. Conservative amino-acid substitutions are lesslikely to affect the functionality of the protein. Amino acids may begrouped according to their shared characteristics. A conservativeamino-acid substitution is a substitution of one amino acid within apredetermined group of amino acids for another amino acid within thesame group, within which the amino acids exhibit similar orsubstantially similar characteristics. Within the meaning of the term“conservative amino acid substitution” as applied herein, one amino acidmay be substituted by another within groups of amino acids characterizedby having

-   -   i) polar side chains (Asp, Glu, Lys, Arg, His, Asn, Gin, Ser,        Thr, Tyr and Cys)    -   ii) non-polar side chains (Gly, Ala, Val, Leu, lie, Phe, Trp,        Pro and Met)    -   iii) aliphatic side chains (Gly, Ala Val, Leu and Ile)    -   iv) cyclic side chains (Phe, Tyr, Trp, His and Pro)    -   v) aromatic side chains (Phe, Tyr and Trp)    -   vi) acidic side chains (Asp and Glu)    -   vii) basic side chains (Lys, Arg and His)    -   viii) amide side chains (Asn and Gin)    -   ix) hydroxyl side chains (Ser and Thr)    -   x) sulfur-containing side chains (Cys and Met)    -   xi) amino acids being monoamino-dicarboxylic acids or        monoamino-monocarboxylic-monoamidocarboxylic acids (Asp, Glu,        Asn and Gin).

A functional homologue within the scope of the present invention is apolypeptide that exhibits at least 50% sequence identity with anaturally occurring form of human GM-CSF, such as at least 60% sequenceidentity, for example at least 70% sequence identity, such as at least75% sequence identity, for example at least 80% sequence identity, suchas at least 85% sequence identity, for example at least 90% sequenceidentity, such as at least 91% sequence identity, for example at least91% sequence identity, such as at least 92% sequence identity, forexample at least 93% sequence identity, such as at least 94% sequenceidentity, for example at least 95% sequence identity, such as at least96% sequence identity, for example at least 97% sequence identity, suchas at least 98% sequence identity, for example 99% sequence identitywith a naturally occurring form of human GM-CSF.

Sequence identity can be calculated using a number of well-knownalgorithms and applying a number of different gap penalties. Anysequence alignment algorithm, such as but not limited to FASTA, BLAST,or GETSEQ, may be used for searching homologues and calculating sequenceidentity. Moreover, when appropriate, any commonly known substitutionmatrix, such as but not limited to PAM, BLOSSUM or PSSM matrices, may beapplied with the search algorithm. For example, a PSSM (positionspecific scoring matrix) may be applied via the PSI-BLAST program.Moreover, sequence alignments may be performed using a range ofpenalties for gap-opening and extension. For example, the BLASTalgorithm may be used with a gap-opening penalty in the range 5-12, anda gap-extension penalty in the range 1-2.

Accordingly, a variant or a fragment thereof according to the inventionmay comprise, within the same variant of the sequence or fragmentsthereof, or among different variants of the sequence or fragmentsthereof, at least one substitution, such as a plurality of substitutionsintroduced independently of one another.

It is clear from the above outline that the same variant or fragmentthereof may comprise more than one conservative amino-acid substitutionfrom more than one group of conservative amino acids as defined hereinabove.

Aside from the twenty standard amino acids and two special amino acids,selenocysteine and pyrrolysine, there are a vast number of “non-standardamino acids” which are not incorporated into protein in vivo. Examplesof nonstandard amino acids include the sulfur-containing taurine and theneurotransmitters GABA and dopamine. Other examples are lanthionine,2-aminoisobutyric acid, and dehydroalanine. Further non-standard aminoare ornithine and citrulline.

Non-standard amino acids are usually formed through modifications tostandard amino acids. For example, taurine can be formed by thedecarboxylation of cysteine, while dopamine is synthesized from tyrosineand hydroxyproline is made by a posttranslational modification ofpraline (common in collagen). Examples of non-natural amino acids arethose listed e.g. in 37 C.F.R. section 1.822 (b)(4), all of which areincorporated herein by reference.

Both standard and non-standard amino acid residues described herein canbe in the “D” or “L” isomeric form.

It is contemplated that a functional equivalent according to theinvention may comprise any amino acid including non-standard aminoacids. In preferred embodiments, a functional equivalent comprises onlystandard amino acids.

The standard and/or non-standard amino acids may be linked by peptidebonds or by non-peptide bonds. The term peptide also embracespost-translational modifications introduced by chemical orenzyme-catalyzed reactions, as are known in the art. Suchpost-translational modifications can be introduced prior topartitioning, if desired. Amino acids as specified herein willpreferentially be in the L-stereoisomeric form. Amino acid analogs canbe employed instead of the 20 naturally occurring amino acids. Severalsuch analogs are known, including fluorophenylalanine, norleucine,azetidine-2-carboxylic acid, S-aminoethyl cysteine, 4-methyl tryptophanand the like.

In one embodiment of the present invention, the GM-CSF variant comprisesa conjugate capable of prolonging half-life of the active ingredient,such as for example albumin or a fatty acid.

Suitable variants will be at least 60% identical, preferably at least70%, and accordingly, variants preferably have at least 75% sequenceidentity, for example at least 80% sequence identity, such as at least85% sequence identity, for example at least 90% sequence identity, suchas at least 91% sequence identity, for example at least 91% sequenceidentity, such as at least 92% sequence identity, for example at least93% sequence identity, such as at least 94% sequence identity, forexample at least 95% sequence identity, such as at least 96% sequenceidentity, for example at least 97% sequence identity, such as at least98% sequence identity, for example 99% sequence identity with thepredetermined sequence of a naturally occurring form of human GM-CSF.

Functional homologues may further comprise chemical modifications suchas ubiquitination, labeling (e.g., with radionuclides, various enzymes,etc.), pegylation (derivatization with polyethylene glycol), or byinsertion (or substitution by chemical synthesis) of amino acids such asornithine, which do not normally occur in human proteins.

In addition to the peptidyl compounds described herein, stericallysimilar compounds may be formulated to mimic the key portions of thepeptide structure and such compounds may also be used in the same manneras the polypeptides of the invention. This may be achieved by techniquesof modelling and chemical designing known to those of skill in the art.For example, esterification and other alkylations may be employed tomodify the amino terminus (N-terminus) of, e.g., a di-arginine peptidebackbone, to mimic a tetrapeptide structure. It will be understood thatall such sterically similar constructs fall within the scope of thepresent invention.

Peptides with N-terminal alkylations and C-terminal esterifications arealso encompassed by the present invention. Functional equivalents alsocomprise glycosylated and covalent or aggregative conjugates formed withthe same molecules, including dimers or unrelated chemical moieties.Such functional equivalents are prepared by linkage of functionalitiesto groups which are found in a fragment that includes any one or both ofthe N- and C-termini, by means known in the art.

The term “fragment thereof” may refer to any portion of the givenamino-acid sequence. Fragments may comprise more than one portion fromwithin the full-length protein, joined together. Suitable fragments maybe deletion or addition mutants. The addition of at least one amino acidmay be an addition of from preferably 2 to 250 amino acids, such as from10 to 20 amino acids, for example from 20 to 30 amino acids, such asfrom 40 to 50 amino acids. Fragments may include small regions from theprotein or combinations of these. The deletion and/or the addition may,independently of one another, be a deletion and/or an addition within asequence and/or at the end of a sequence.

Deletion mutants suitably comprise at least 20 or 40 consecutive aminoacid and more preferably at least 80 or 100 consecutive amino acids inlength. Accordingly, such a fragment may be a shorter sequence takenfrom the sequence of human GM-CSF comprising at least 20 consecutiveamino acids, for example at least 30 consecutive amino acids, such as atleast 40 consecutive amino acids, for example at least 50 consecutiveamino acids, such as at least 60 consecutive amino acids, for example atleast 70 consecutive amino acids, such as at least 80 consecutive aminoacids, for example at least 90 consecutive amino acids, such as at least95 consecutive amino acids, such as at least 100 consecutive aminoacids, such as at least 105 amino acids, for example at least 110consecutive amino acids, such as at least 115 consecutive amino acids,for example at least 120 consecutive amino acids, wherein said deletionmutants preferably has at least 75% sequence identity, for example atleast 80% sequence identity, such as at least 85% sequence identity, forexample at least 90% sequence identity, such as at least 91% sequenceidentity, for example at least 91% sequence identity, such as at least92% sequence identity, for example at least 93% sequence identity, suchas at least 94% sequence identity, for example at least 95% sequenceidentity, such as at least 96% sequence identity, for example at least97% sequence identity, such as at least 98% sequence identity, forexample 99% sequence identity with a naturally occurring form of humanGM-CSF.

It is preferred that functional homologues of GM-CSF comprise at most500, more preferably at most 400, even more preferably at most 300, yetmore preferably at most 200, such as at most 175, for example at most160, such as at most 150 amino acids, for example at most 144 aminoacids.

There are two known variants of human GM-CSF: a T1151 substitution invariant 1 and a I117T substitution in variant 2. Accordingly, in oneembodiment of the invention, a functional homologue of GM-CSF comprisesa sequence with high sequence identity to human GM-CSF NO: 1 or any ofthe splice variants.

Analogues of GM-CSF are, for example, described in U.S. Pat. Nos.5,229,496, 5,393,870, and 5,391,485. Such analogues are also functionalequivalents comprised within the present invention.

In one embodiment, GM-CSF is used according to the present invention inhomo- or heteromeric form. Homo- and heteromeric forms of GM-CSF maycomprise one or more GM-CSF monomers or functional homologous of GM-CSFas defined herein above. Homo- and heteromers include dimers, trimers,tetramers, pentamers, hexamers, heptamers, octamers, nonamers anddecamers.

In one embodiment, a homodimer, trimer or tetramer of GM-CSF is used.

The amino-acid sequence of the precursor (including the signal peptide)form of GM-CSF of Homo sapiens (SEQ ID NO:1) is:

MWLQSLLLLG TVACSISAPA RSPSPSTQPW EHVNAIQEARRLLNLSRDTA AEMNETVEVI SEMFDLQEPT CLQTRLELYKQGLRGSL TKL KGPLTMMASH YKQHCPPTPE TSCATQIITF ESFKENLKDF LLVIPFDCWE PVQE.

The amino-acid sequence of the corresponding mature protein (SEQ ID NO:2) is:

APARSPSPST QPWEHVNAIQ EARRLLNLSR DTMEMNETVEVISEMFDLQ EPTCLQTRLE LYKQGLRGSL TKLKGPLTMMASHYKQHCPP TPETSCATQI ITFESFKENL KDFLLVIPFD CWEPVQE

Functional homologues of a naturally occurring form of human GM-CSFaccording to the present invention may be commercially available, e.g.sargramostim (Leukine®; Immunex, Seattle, Wash., USA).

Recombinant Production of GM-CSF

GM-CSF or functional variants or homologues thereof can be produced invarious ways, such as isolation from for example human or animal serumor from expression in cells, such as prokaryotic cells, yeast cells,insect cells, mammalian cells or in cell-free systems.

In one embodiment of the invention, GM-CSF is produced recombinantly byhost cells. Thus, in one aspect of the present invention, GM-CSF isproduced by host cells comprising a first nucleic acid sequence encodingthe GM-CSF operably associated with a second nucleic acid sequencecapable of directing expression in said host cells. The second nucleicacid sequence may thus comprise or even consist of a promoter that willdirect the expression of protein of interest in said cells. A skilledperson will be readily capable of identifying useful second nucleic acidsequence for use in a given host cell.

The process of producing a recombinant GM-CSF in general comprises thesteps of

-   -   providing a host cell    -   preparing a gene expression construct comprising a first nucleic        acid sequence encoding the GM-CSF operably linked to a second        nucleic acid sequence capable of directing the expression of        said protein of interest in the host cell    -   transforming the host cell with the construct    -   cultivating the host cell, thereby obtaining expression of the        GM-CSF.

The recombinant GM-CSF thus produced may be isolated by any conventionalmethod, such as any of the methods for protein isolation describedherein below. The skilled person will be able to identify suitableprotein isolation steps for purifying the GM-CSF.

In one embodiment of the invention, the recombinantly produced GM-CSF isexcreted by the host cells. When the GM-CSF is excreted, the process ofproducing a recombinant protein of interest may comprise the steps of

-   -   providing a host cell    -   preparing a gene expression construct comprising a first nucleic        acid sequence encoding the GM-CSF operably linked to a second        nucleic acid sequence capable of directing the expression of        said protein of interest in said host cell    -   transforming said host cell with the construct    -   cultivating the host cell, thereby obtaining expression of the        GM-CSF and secretion of the GM-CSF into the culture medium    -   thereby obtaining culture medium containing the GM-CSF.

The composition comprising GM-CSF and nucleic acids may thus in thisembodiment of the invention be the culture medium or a compositionprepared from the culture medium.

In another embodiment of the invention, said composition is an extractprepared from animals, parts thereof or cells or an isolated fraction ofsuch an extract.

In an embodiment of the invention, the GM-CSF is recombinantly producedin vitro in host cells and isolated from cell lysate, cell extract orfrom tissue culture supernatant. In a more preferred embodiment, theGM-CSF is produced by host cells that are modified in such a way thatthey express the relevant GM-CSF. In an even more preferred embodimentof the invention, said host cells are transformed to produce and excretethe relevant GM-CSF.

Pharmaceutical compositions according to the present invention maycomprise GM-CSF or functional variants or homologues thereof at aconcentration of 1 μg/mL to 10 mg/mL, more preferably 5 μg/mL to 500μg/mL, and even more preferably 10 μg/mL to 200 μg/mL.

Fosfomycin Preparations

The fosfomycin preparations that fall within the scope of the presentinvention are compounds and salts of compounds that comprise thestructure (−)(IR, 2 S)-1,2-epoxypropylphosphonic acid, systematic name[(2R,3S)-3-methyloxiran-2-yl]phosphonic acid, formula weight 138.1 Da.Although the free acid form of this structure is used as a basis forcalculating the effective amounts and concentrations of fosfomycin, thefree acid is unstable and the antibiotic is presented for clinical useas an inorganic or organic salt. Non-limiting examples of inorganiccounter-ions of fosfomycin salts within the scope of the presentinvention are sodium, calcium, potassium, lithium, ammonium, magnesium.Non-limiting examples of organic counter-ions of fosfomycin salts withinthe scope of the present invention are trometamol (also known astromethamine or tris, systematic name2-amino-2-hydroxymethyl-propane-1,3-diol), phenylethylamine, and a largenumber of other biocompatible organic amines. The principal forms offosfomycin in current use that come within the scope of this inventionare:

-   -   i) Fosfomycin disodium, formula weight 182.0 Da, pH of 5%        solution 9.0-10.5. This salt is highly soluble in water,        irritant to the stomach, and is principally used for intravenous        injection.    -   ii) Fosfomycin calcium monohydrate, formula weight 194.1 Da, pH        of 0.4% solution 8.1-9.6. This salt is sparingly soluble in        water but is less irritating to the stomach and is used for oral        treatment. Its bioavailability may be as low as 12% (Bergan T,        1990).    -   iii) Fosfomycin trometamol, formula weight 259.2 Da, pH of 5%        solution 3.5-5.5. This salt is highly soluble in water, is well        tolerated when given orally and is used especially as a        single-dose oral treatment for lower urinary tract infection,        showing a bioavailability of about 40%.

Common to these preparations is that they are stable for at least 3years as dry powders at 25° C., but show a pH-dependent instability inaqueous solution, at 25° C. losing 10% of fosfomycin activity within 10h at pH 3, 2 months at pH 6.5 and 2 years at pH 9.75. Reduced stabilityat low pH must be taken into in account when formulating aqueouscompositions for topical application with a suitable shelf life.

Combination with Other Antimicrobial Agents

A composition of the present invention may also contain one or moreadditional antimicrobial agents to potentiate its bactericidal action onimportant pathogens such as Staphylococcus aureus or Pseudomonasaeruginosa, prevent the overgrowth of any strains that developresistance on prolonged treatment, or broaden the antimicrobial spectrumto include non-bacterial pathogens, including but not limited to fungi.Non-limiting examples of antimicrobial agents that may be included inthe composition are: fusidic acid or sodium fusidate, penicillins,cephalosporins, aminoglycosides, macrolides, vancomycin, lincomycin,clindamycin, fluoroquinolones, mupirocin, bacitracin, polymyxin B,gramicidins, metronidazole, clotrimazole, ketoconazole and nystatin.Particularly suitable for inclusion are agents which show a synergicbactericidal action with fosfomycin, non-limiting examples of which are:penicillin, ampicillin, carbenicillin, methicillin, oxacillin,mezlocillin, piperacillin, aztreonam, imipenem, cephalexin, cephalothin,cefamandole, cefoxitin, cefmetazole, cefotaxime, cefazolin,cefoperazone, cefsulodine, ceftadizime, cefepime, streptomycin,gentamicin, kanamycin, netilmicin, tobramycin, amikacin, erythromycin,midecamycin, vancomycin, lincomycin, clindamaycin, teicoplanin,daptomycin, ciprofloxacin, ofloxazine, levofloxazin, pefloxacin,sparfloxacin. Certain antibiotics are incompatible with fosfomycin inaqueous solution, such as ampicillin, methampicillin, cephaloridine,cephalothin, streptomycin, gentamicin, kanamycin, a feature that maymake them unsuitable for aqueous compositions within the scope of thisinvention.

In one embodiment, the additional antibiotic or antibacterial agent isselected from the list of arbekacin, aztreonam, cefoxitin, cefoperazone,cephalexin, clindamycin, flucloxacillin, meropenem, metronidazole,mezlocillin, or vancomycin. The selection of clindamycin or meropenem ormetronidazole is particularly intended for the treatment of woundssuspected or known through appropriate bacterial culture to be infectedwith anaerobic bacteria including Bacteroides species.

In another embodiment, the additional antibiotic is at least acombination of aztreonam and arbekacin. This is particularly intendedfor the treatment of wounds suspected or known through appropriatebacterial culture to be infected with Pseudomonas aeruginosa.

As all the above compounds are known to the skilled person, it will notpresent an undue burden to the skilled person to choose from the list ofcompounds which compound(s) to combine with the GM-CSF and fosfomycintreatment on the basis of test data on the identity of the infectiveorganisms that are to be treated.

Further Ingredients of the Pharmaceutical Composition

Vitamin A and anti-oxidant agents may further have a stimulating effecton the tissue during the process of healing. In one embodiment of thepresent invention, the composition as defined herein further comprisesvitamin A and/or an anti-oxidant agent, non-limiting examples of whichare: vitamin E (in the form of alpha-tocopherol), ubiquinone, idebenone,carotenoids such as lycopene, ascorbic acid or ascorbates, andnicotinamide.

Dose

By “effective amount” of the pharmaceutical compositions of the presentinvention is meant a dose, which, when administered to a subject in needthereof, achieves a concentration which has a beneficial biologicaleffect, i.e. by treatment, prevention or alleviation of irritation orlesion such as wounds, ulcers and other lesions of the skin, mucosalmembranes or connective tissue of the body. Such an effective amount maybe determined by a patient's attending physician or veterinarian and isreadily ascertained by one of ordinary skill in the art. Factors whichinfluence what a therapeutically effective amount will be include thefollowing:

-   -   i) The specific activity of the therapeutic agent being used,    -   ii) The type of lesion (mechanical or thermal, full or partial        thickness, etc.)    -   iii) The size of the lesion    -   iv) The depth of the lesion (if full thickness)    -   v) The presence of infection and the type of infection    -   vi) The time elapsed since the infliction of the injury        infliction    -   vii) The existence of other disease states    -   viii) The age, physical condition, and nutritional status of the        patient

Other medication that the patient may be receiving will affect thedetermination of the therapeutically effective amount of the therapeuticagent to administer.

Whereas the effective amounts and dosages of the ingredients of the apharmaceutical composition are determined in relation to body weight orbody surface area for systemic treatments, for topical application of acomposition to wounds and ulcers, the effective amounts and dosages aremore appropriately expressed in terms of the area of wound or ulcer tobe treated, e.g. expressed in square centimeters, provided that thesystemic absorption of the active ingredients does not lead to anadversely high dosage for the patient.

The effective amount of GM-CSF or a functional variant or homologuethereof, for topical application to a wound or ulcer may be from 1microgram (μg) to 100 μg per square centimeter per day, such as in therange of 2 μg to 80 μg per square centimeter per day, and especially inthe range of 5 μg to 50 μg per square centimeter per day. The effectiveamount is expressed in terms of the amount to be given of a fullyfunctional homologue of human GM-CSF such as molgramostim and isadjusted according to functional activity of the homologue used.

In practical terms, a pharmaceutical composition of the presentinvention comprises GM-CSF or a fragment or variant thereof at aconcentration in the range of 1 μg/mL (or μg/g) to 10 mg/mL (or mg/g),such as in the range of 5 μg/mL (or μg/g) to 500 μg/mL (or μg/g), orsuch as in the range of 10 μg/mL (or μg/g) to 200 μg/mL (or μg/g).

The effective amount of a suitable salt of fosfomycin for topicalapplication to a wound or ulcer may be from 10 mg to 1000 μg per squarecentimeter per day, such as in the range of 20 μg to 800 μg per squarecentimeter per day, and especially in the range of 50 μg to 500 μg persquare centimeter per day. The effective amount is expressed in terms ofthe content of fosfomycin free acid in the preparation used.

In practical terms, a pharmaceutical composition of the presentinvention comprises a fosfomycin salt at a concentration in the range of100 μg/mL (or μg/g) to 30 mg/mL (or mg/mL), such as in the range of 250μg/mL (or μg/g) to 10 mg/mL (or mg/g) in terms of fosfomycin free acid.

The effective amount of an additional antimicrobial agent for topicalapplication is known in the art and may be added to the pharmaceuticalcompositions of the present invention in an amount that is adjusted sothat when an effective amount of GM-CSF and fosfomycin is given, aneffective amount of the additional antimicrobial agent is also given. Inpractice, this means that fusidic acid, for example, will be added tothe composition in a similar amount to that of fosfomycin, whiletobramycin, for example, will be added in an amount that is between 1%and 10% of the amount of fosfomycin.

The effective daily dose is preferably administered once a day, but maybe administered in divided doses twice a day, three times a day, fourtimes a day, five times a day or six times a day.

Duration of dosing will typically range from 1 day to about 4 months,such as in the range of 1 day to 2 days, 2 days to 3 days, 3 days to 4days, 4 days to 5 days, 5 days to 6 days, or in the range of 1 week to 2weeks, 2 weeks to 3 weeks, 3 weeks to 4 weeks, or in the range of 1month to 2 months, 2 months to 3 months, 3 months to 4 months, as longas the lesion remains unhealed.

The transformation of a resting macrophage into a fully immunocompetentdendritic cell after in vitro incubation of macrophages with GM-CSFtakes approximately 10 days. In one embodiment, a duration of a dose hasthe length allowing for said a transformation, thus the duration can bein the range of 7 days to 14 days, such as 8 days to 12 days, forexample 8 days, or 9 days, or 10 days, or 11 days, or 12 days.

A dose regime may alternate between periods of administration of thepharmaceutical composition according to the present invention andperiods with no administration (a pause in treatment). A period with apause of treatment in such a dose regime may last for 5 days to 10 days,for example 5 days, or 6 days, or 7 days, or 8 days, or 9 days, or forexample 10 days or more, for example 1 to 4 months.

Examples of dosage regimes may include a cycle of 10 days of treatmentwith the pharmaceutical composition according to the present inventionand 7 days' pause of treatment. The pause of treatment may be prolongedto 2 or 3 weeks or more, up to 6 weeks, if alternative treatment with apreparation that does not contain antibiotic is substituted. This is toprevent the generation of antibiotic-resistant organisms in theindividual wound.

The conversion of resting macrophages into dendritic cells may beboosted by repeating a dosage regime. Thus dosage regimes can berepeated one, two, three, four, five or more times in order to obtain aneffective treatment.

In one embodiment, a dosage regime is repeated, such as once, two times,three times or more times, for example repeated for the rest of thelifespan of a subject in need.

In another embodiment, patients are treated with a dosage regime of 10days treatment with pharmaceutical composition according to the presentinvention, followed by a pause of 7-20 days in said treatment andsubsequently repeating the dosage regime 2-3 or more times.

Formulations

The pharmaceutical composition of the present invention may be in theform of a powder, dusting powder spray, paste, ointment, lotion, gel,cream, salve, emulsion, suspension, solution, spray, sponge, strip,plaster, pad, dressing, or formulated in an ostomy plate. The activeingredients of the composition may be suspended as a micronized powderin non-aqueous media or dissolved in aqueous media.

A dusting powder is a preferred formulation, as the active ingredientsof the composition are compatible with each other and show long-termstability at room temperature in the dry powder form. The formulationmay contain powder additives such as starch stearate, cellulose,lactose, zinc oxide, silicon dioxide, magnesium carbonate, talc or clay.

An ointment comprising hydrocarbon gels without an aqueous component isalso a preferred formulation, as the fosfomycin in micronized form isnot subject to degradation by hydrolysis in the presence of water atsub-alkaline pH levels, and will have a suitably long shelf life. Theointment may be made into a paste by the addition of powders such asstarch, zinc oxide, talc or titanium dioxide.

Example 2 gives the composition of an ointment to provide an effectivedose of GM-CSF and fosfomycin in a single daily application.

Example 3 gives the composition of an ointment to provide an effectivedose of GM-CSF and fosfomycin together with clindamycin in a singledaily application to provide a strong action against anaerobic bacteriaand also against methicillin-resistant Staphylococcus aureus (MRSA).

Example 4 gives the composition of an ointment to provide an effectivedose of GM-CSF and fosfomycin together with arbekacin and aztreonam in asingle daily application to provide a strong action against Pseudomonasaeruginosa.

Aqueous media such as gels, creams, lotions, solutions and suspensionscan also be used, but will require that the pH be adjusted to a valuebetween pH 7 and pH 10, preferably to at least pH 8, to ensure areasonable stability of the fosfomycin component and thus a suitableshelf life for the formulation. The pH of the aqueous medium can beadjusted by means of low concentrations of suitable biocompatiblebuffering ingredients, non-limiting examples being tromethamine, sodiumcarbonate and bicarbonate, as well as sodium dihydrogen phosphate anddisodium hydrogen phosphate. Tonicity adjusting agents, such as forexample sodium chloride, potassium chloride, or calcium chloride, mayalso be added.

The compositions according to the present invention may be formulatedfor improved penetration and efficacy of the active ingredients in theirpassage over the transmucosal barrier or epidermis. Improved passageover the transmucosal barrier may be obtained by a formulation which iscapable of adhering to the mucosa, epidermis, or wound surface. This isan intrinsic characteristic of the ointment formulation proposed, butspecial agents which improve penetration may be added, non-limitingexamples of which are propylene glycol, polyethylene glycol,dimethylsulfoxide, 1-decyl dimethylsulfoxide, N-methylpyrrolidone,diethyl toluamide, isopropyl myristate, isopropyl palmitate and estersof oleic acid.

Formulations according to the present invention may comprisepharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, micelles, microcapsules, nanoparticles or thelike. The GM-CSF component may, for example, be formulated in a liposomewith an outer fatty layer with a core of water phase in which the GM-CSFcomponent is dissolved. The lipid layer of such formulations overcomesthe penetration barrier of the epidermis or mucous membrane.

Conventional liposomes are typically composed of phospholipids (neutralor negatively charged) and/or cholesterol. The liposomes are vesicularstructures based on lipid bilayers surrounding aqueous compartments.They can vary in their physicochemical properties such as size, lipidcomposition, surface charge and number and fluidity of the phospholipidsbilayers. The most frequently used lipid for liposome formation are:1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC),1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC),1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC),1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC),1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE),1,2-dimyristoyl-sn-glycero-3-phosphate (monosodium salt) (DMPA),1,2-dipalmitoyl-sn-glycero-3-phosphate (monosodium salt) (DPPA),1,2-dioleoyl-sn-glycero-3-phosphate (monosodium salt) (DOPA),1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodium salt)(DMPG), 1,2-dipalmitoyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (sodiumsalt) (DPPG), 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1-glycerol)](sodium salt) (DOPG), 1,2-dimyristoyl-sn-glycero-3-[phospho-1-serine](sodium salt) (DMPS), 1,2-dipalmitoyl-sn-glycero-3-[phospho-1-serine)(sodium salt) (DPPS), 1,2-dioleoyl-sn-glycero-3-[phospho-1-serine](sodium salt) (DOPS),1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-(glutaryl) (sodium salt)and 1,1′,2,2′tetramyristoyl cardiolipin (ammonium salt). Formulationscomposed of DPPC in combination with other lipids or modifiers ofliposomes are preferred, e.g. in combination with cholesterol and/orphosphatidylcholine.

A useful way of producing liposomes is to attach hydrophilic polymerpolyethylene glycol (PEG) covalently to the outer surface of theliposome. Some of the preferred lipids are:1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n-[methoxy(polyethyleneglycol)-2000] (ammonium salt),1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-n[methoxy(polyethyleneglycol)-5000](ammonium salt), 1,2-dioleoyl-3-trimethylammonium-propane (chloridesalt) (DOTAP).

Possible lipids applicable for liposomes are supplied by e.g. Avanti,Polar Lipids, Inc., Alabaster, Ala., USA. Additionally, the liposomesuspension may include lipid-protective agents which protect lipidsagainst free-radical and lipid-peroxidative damage on storage.Lipophilic free-radical quenchers, such as alpha-tocopherol andwater-soluble iron-specific chelators, such as ferrioxamine, arepreferred.

Several methods are available for preparing liposomes, as described in,e.g., Szoka F et al. (1980), U.S. Pat. Nos. 4,235,871, 4,501,728 and4,837,028, all of which are incorporated herein by reference. Anothermethod produces multi-lamellar vesicles of heterogeneous sizes. In thismethod, the vesicle-forming lipids are dissolved in a suitable organicsolvent or solvent system and dried under vacuum or an inert gas to forma thin lipid film. If desired, the film may be re-dissolved in asuitable solvent, such as tertiary butanol, and then lyophilized to forma more homogeneous lipid mixture which is in a more easily hydratedpowder-like form. This film is covered with an aqueous solution of thetargeted drug and the targeting component and allowed to hydrate,typically over a 15-60 minute period with agitation. The sizedistribution of the resulting multi-lamellar vesicles can be shiftedtoward smaller sizes by hydrating the lipids under more vigorousagitation conditions or by adding solubilizing detergents such asdeoxycholate.

To ensure adequate shelf-life of the components of the dermatologicalformulation, especially that of fosfomycin, which is unstable in aqueousmedia at physiological pH, while at the same time providing aformulation that is acceptable to the patient in terms of irritation atthe site of application, an embodiment of the invention comprises theprovision of the active components in a kit as separate parts to bemixed shortly before use. The kit may further comprise a medium intowhich the components can be mixed prior to the topical application tothe wound. The medium may contain, in addition to other ingredients,buffering agents such as those mentioned above to ensure that the pH ofthe formulation is appropriate for dermatological use once all theingredients have been mixed.

Indications

The present invention provides pharmaceutical compositions for use inthe treatment of wounds, ulcers, sores, burns or other lesions of theskin, mucosal membranes or connective tissue of the body, which may beacute or chronic. Such lesions may be caused by a broad spectrum ofevents and/or may be associated with other diseases. The lesions to betreated include those associated with incision, laceration, abrasion,blister, hematoma, puncture, penetration, gunshot, electricity,irradiation, chemical, trauma, crush, bite, burn, frost, surgery,primary cancer or metastasis, benign tumor, acne, infections such asbacterial infection (which may be combined with fungal or viral ofparasitic infection), lesions associated with decreased circulation ofblood, such as leg ulcers and foot ulcers associated with venousinsufficiency or arterial insufficiency, decubitus ulcers, pressuresores or bedsores, and lesions associated with diabetes mellitus.

Chronic wounds, now generally called “non-healing wounds”, or lesions,wounds or ulcers arise when a wound fails to follow an appropriatetimely healing process to achieve the normal sustained and stableanatomic and functional integrity of the healed tissue. Generallyspeaking, a skin lesion which has failed to make substantial progresstowards healing within a period of three months, or which has becomestable in a partially healed state for more than three months, could becategorized as a chronic or “non-healing” wound. This general definitionis not universally applicable, as the age and fitness of the patient, aswell as other factors such as diseases or disorders suffered by thepatient (for example, circulatory disorders), can significantly lengthenthe normal healing process. In such circumstances a skin lesion which isunhealed after six months can be categorized as a “non-healing” wound.

A “non-healing” wound or chronic skin lesion is ulcerous when itinvolves focal loss of epidermis and at least part of the dermis.Chronic ulcerous skin lesions are usually accompanied by other symptomsapart from the failure of the normal healing process. Typicalaccompanying signs and symptoms include one or more of the following:pain, exudation, bad smell, excoriation, wound spreading, tissuenecrosis, irritation and hyperkeratosis. Such symptoms can be extremelydebilitating and embarrassing for patients, and can seriously harm thepatient's quality of life. In severe cases, they can necessitate theamputation of limbs or even cause death.

Malignant or pre-malignant chronic ulcerous skin lesions may arise inconnection with a primary cancer of the skin, or with a metastasis tothe skin from a local tumor or from a tumor at a distant site. They maybe draining or non-draining. They may, for example, take the form of acavity, an open area on the surface of the skin, skin nodules, or anodular growth extending from the surface of the skin.

The pharmaceutical compositions of the present invention are useful fortreatment of all the above-mentioned non-healing wounds or chroniculcerous skin lesions, and thus reduce or prevent one or more symptomsaccompanying such lesions.

EMBODIMENTS

-   -   1. A pharmaceutical composition comprising        -   a. granulocyte-macrophage colony-stimulating factor (GM-CSF)            or a fragment or variant thereof, and        -   b. osfomycin in the form of an inorganic or organic salt            thereof    -   2. The pharmaceutical composition according to embodiment 1        comprising one or more additional antibiotic or antimicrobial        agents.    -   3. The composition according to embodiment 2, wherein the one or        more additional antibiotic or antimicrobial agent is selected        from the list of fusidic acid, penicillins, cephalosporins,        aminoglycosides, macrolides, vancomycin, lincomycin,        clindamycin, fluoroquinolones, mupirocin, bacitracin, polymyxin        B, gramicidins, metronidazole, clotrimazole, ketoconazole and        nystatin.    -   4. The composition according to embodiment 2 or 3, wherein the        one or more additional antibiotic or antimicrobial agent is        selected from the list of penicillin, ampicillin, carbenicillin,        methicillin, oxacillin, flucloxacillin, mezlocillin,        piperacillin, aztreonam, imipenem, cephalexin, cephalothin,        cefamandole, cefoxitin, cefmetazole, cefotaxime, cefazolin,        cefoperazone, cefsulodine, ceftadizime, cefepime, streptomycin,        gentamicin, kanamycin, netilmicin, tobramycin, amikacin,        erythromycin, midecamycin, vancomycin, lincomycin, clindamaycin,        teicoplanin, daptomycin, ciprofloxacin, ofloxazine,        levofloxazin, pefloxacin, sparfloxacin, ceftriaxone, arbekacin,        or vancomycin.    -   5. The composition according to any one of the preceding        embodiments, wherein the wound or lesion is infected with        staphylococcus such as Staphylococcus aureus or pseudomonas such        as Pseudomonas aeruginosa.    -   6. The composition according to any one of the preceding        embodiments, wherein the composition is not in an aqueous        solution, and wherein the additional antibiotic or microbial        agent is any one of ampicillin, methampicillin, cephaloridine,        cephalothin, streptomycin, gentamicin, or kanamycin.    -   7. The composition of any one of the preceding embodiments,        wherein the composition further comprises one or more of the        following: vitamin A, an antioxidant agent, such as one or more        of the following: vitamin E (in the form of alphatocopherol),        ubiquinone, idebenone, carotenoids in example lycopene, ascorbic        acid or ascorbates, or nicotinamide.    -   8. The pharmaceutical composition according to any one of the        previous embodiments, wherein the composition is for the        treatment, alleviation or accelerating the healing of a lesion        such as a wound, ulcer, sore or burn of any one of the skin,        mucosal membranes or connective tissue underlying the lesion.    -   9. The pharmaceutical composition according to anyone of the        previous embodiments, wherein the lesion is chronic.    -   10. The pharmaceutical composition according to any one of        embodiments 1-11, wherein the lesion is acute.    -   11. The pharmaceutical composition according to any one of the        previous embodiments, wherein the lesion is associated with        colonization or infection by a bacterium, fungus, virus, or        parasite.    -   12. The composition according to any one of the previous        embodiments, wherein the lesion is infected with bacteria from        any one of the genera Staphylococcus, Streptococcus, Neisseria,        Escherichia, Proteus, Serratia, Salmonella, Shigella,        Pseudomonas, Haemophilus, and Vibrio, Proteus spp., Klebsiella        and Enterobacter spp., Peptostreptococcus (including        Peptoniphilus, Finegoldia and Anaerococcus) and Fusobacterium.    -   13. The composition according to any one of the previous        embodiments, wherein the active components are provided in a kit        as separate parts to be mixed shortly before use.    -   14. The kit according to embodiment 13, wherein the kit further        comprises a medium into which the components can be mixed prior        to the topical application to the wound.    -   15. The pharmaceutical composition according to any one of the        previous embodiments, wherein the lesion is associated with        diabetes mellitus.    -   16. The pharmaceutical composition according to any one of the        previous embodiments, wherein the lesion is associated with        decreased circulation of blood, such as venous leg ulcers,        venous foot ulcers, arterial leg ulcers, arterial foot ulcers,        and decubitus ulcers.    -   17. The pharmaceutical composition according to any one of the        previous embodiments formulated for topical application as a        powder, paste, ointment, lotion, gel, cream, salve, emulsion,        suspension, solution, spray, sponge, strip, plaster, pad,        dressing, or formulated in an ostomy plate.    -   18. The composition according to any one of the previous        embodiments, wherein GM-CSF and fosfomycin is for topical        administration, and the additional antimicrobial drug is for        non-topical administration.    -   19. The pharmaceutical composition according to any one of        embodiments 1 to 14 wherein the GM-CSF is in a liposomal or        micelle or microcapsule or nanoparticle formulation.    -   20. The pharmaceutical composition according to any one of the        preceding embodiments wherein the GM-CSF variant is at least 70%        identical to SEQ ID NO:1 or 2.    -   21. The pharmaceutical composition according to any one of the        preceding embodiments wherein the GM-CSF fragment comprises at        least 50 contiguous amino acid residues of any one of SEQ ID        NO:1 or 2.    -   22. The pharmaceutical composition according to embodiment 21,        wherein the fragment is at least 70% identical to SEQ ID NO:1 or        2 in the range of overlap.    -   23. The pharmaceutical composition according to any one of the        preceding embodiments, comprising GM-CSF or a fragment or        variant thereof at a concentration of 1 μg/mL to 10 mg/mL.    -   24. The pharmaceutical composition according to any one of the        preceding embodiments, comprising GM-CSF or a fragment or        variant thereof at a concentration of 5 μg/mL to 500 μg/mL.    -   25. The pharmaceutical composition according to any one of the        preceding embodiments, comprising GM-CSF or a fragment or        variant thereof at a concentration of 10 μg/mL to 200 μg/mL.    -   26. The pharmaceutical composition according to any one of the        preceding embodiments, comprising a fosfomycin salt at a        concentration of 100 μg/mL to 10 mg/mL in terms of fosfomycin        free acid.    -   27. The pharmaceutical composition according to any one of the        preceding embodiments, comprising a fosfomycin salt at a        concentration pf 250 μg/mL to 1 mg/mL in terms of fosfomycin        free acid.    -   28. The pharmaceutical composition according to embodiments 17        and 18, wherein the fosfomycin salt is fosfomycin disodium or        fosfomycin calcium or fosfomycin trometamol, also known as        fosfomycin tromethamine.    -   29. The pharmaceutical composition according to any one of the        preceding embodiments, further comprising one or more additional        antibiotic or antimicrobial agents.    -   30. The pharmaceutical composition according to any one of the        preceding embodiments further comprising vitamin A and/or an        anti-oxidant agent.    -   31. The pharmaceutical composition according to any one of the        preceding embodiments, wherein the GM-CSF variant comprises a        conjugate capable of prolonging the half-life of the GM-CSF.    -   32. The pharmaceutical composition according to the preceding        embodiment, wherein the conjugate capable of prolonging        half-life of the GM-CSF variant is albumin or a fatty acid.    -   33. The pharmaceutical compositions according to any one of the        preceeding claims are also useful I in methods of treatment.

EXAMPLES

The following non-limiting examples further illustrate the presentinvention.

Example 1: Sequences

SEQ ID NO: 1 - Human GM-CSF precursor >sp|P04141|CSF2_HUMAN Granulocyte-macrophagecolony-stimulating factor OS = Homo sapiensMWLQSLLLLGTVACSISAPARSPSPSTQPWEHVNAIQEARRLLNLSRDTMEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATOI ITFESFKEN LKDFLL VI PFDCWEPVQESEQ ID NO: 2 - mature human GM-CSF >sp|P04141|18-144APARSPSPSTQPWEHVNAIQEARRLLNLSRDTMEMNETVEVISEMFDLQEPTCLQTRLELYKQGLRGSLTKLKGPLTMMASHYKQHCPPTPETSCATQIITFESFKENLKDFLLVIPFDCWEPVQE

Example 2: Ointment for Treating Wounds, Ulcers, Sores or Burns of theSkin

Molgramostim 0.5 mg 0.05%  micronized Fosfomycin trometamol 5 mg 0.5%micronized Chlorbutanol, anhydrous 5 mg 0.5% Mineral oil 50 mg  5% WhitePetrolatum to 1 g to 100%

This composition may be especially suitable for leg ulcers due to venousinsufficiency, which do not show clinical signs of gross bacterialinfection.

Example 3: Ointment for Treating Wounds, Ulcers, Sores or Burns of theSkin, which Contains a Further Antibiotic in Addition to Fosfomycin

Molgramostim 0.5 mg 0.05%  micronized Fosfomycin trometamol 5 mg 0.5%micronized Clindamycin phosphate 10 mg 1.0% micronized Chlorbutanol,anhydrous 5 mg 0.5% Mineral oil 50 mg  5% White Petrolatum to 1 g to100%

This composition may be especially suitable for treating wounds andulcers in which infection with anaerobic bacteria is suspected orverified by anaerobic bacterial culture. It may also be suitable forwounds infected with methicillin-resistant Staphylococcus aureus (MRSA)verified by culture to be sensitive to clindamycin.

Example 4: Ointment for Treating Wounds, Ulcers, Sores or Burns of theSkin, which Contains a Further Two Antibiotics in Addition to Fosfomycin

Molgramostim 0.5 mg 0.05%  micronized Fosfomycin trometamol 5 mg 0.5%micronized Arbekacin sulfate 2 mg 0.2% micronized Aztreonam 5 mg 0.5%micronized Chlorbutanol, anhydrous 5 mg 0.5% Mineral oil 50 mg  5% WhitePetrolatum to 1 g to 100%

This composition may be especially suitable for treating wounds andulcers in which infection with Pseudomonas aeruginosa is suspected orverified by bacterial culture.

REFERENCES

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1. A pharmaceutical composition comprising a. granulocyte-macrophagecolony-stimulating factor (GM-CSF) or a fragment or variant thereof, andb. fosfomycin in the form of an inorganic or organic salt thereof.
 2. Amethod of accelerating the healing of a bacterially infected lesioncomprising a wound, ulcer, sore or burn of the skin, mucosal membranesor connective tissue underlying the lesion, comprising: topicallyapplying a pharmaceutical composition containing as active ingredients:i) granulocyte-macrophage colony-stimulating factor (GM-CSF) in the formof molgramostim or sargramostim, and ii) fosfomycin calcium to a subjectthat has said bacterially infected lesion, said composition beingapplied to the lesion as a dry powder.
 3. The method according to claim2 wherein the lesion is chronic.
 4. The method according to claim 2wherein the lesion is acute.
 5. The method according to claim 2, whereinthe lesion is associated with diabetes mellitus.
 6. The method accordingto claim 2, wherein the lesion is associated with a decreasedcirculation of blood, a venous leg ulcer, a venous foot ulcer, anarterial leg ulcer, an arterial foot ulcer, or a decubitus ulcer.
 7. Themethod according to claim 2, wherein the pharmaceutical compositioncontains GM-CSF at a concentration of 1 μg/g to 10 mg/g.
 8. The methodaccording to claim 2, wherein the pharmaceutical composition furthercomprises one or more additional antibiotic or antimicrobial agents. 9.The method according to claim 2, wherein the lesion is colonized by abacterium, fungus, virus, or parasite.
 10. The method according to claim2, wherein the pharmaceutical composition comprises GM-CSF at aconcentration of 5 μg/g to 500 μg/g.
 11. The method according to claim2, wherein the pharmaceutical composition comprises GM-CSF at aconcentration of 10 μg/g to 200 μg/g.